This revised grant application focuses on the role of the endogenous gaseous biological mediator hydrogen sulfide (H2S) in colorectal cancer. Based on multiple lines of novel data, we have developed the novel hypothesis that selective upregulation of cystathionine--synthase (CBS) and the subsequent production of H2S in colonic cancer cells serves as a pro-survival factor by stimulating tumor cell bioenergetics, growth, proliferation, migration and invasion. In order to explore this concept, we will pursue the following Aims: #1. To characterize the molecular mechanisms by which the CBS/H2S axis promotes tumor cell bioenergetics and tumor cell proliferation; #2. To determine the molecular mechanisms by which the CBS/H2S axis promotes tumor cell migration, invasion and metastasis; and #3. To evaluate the effect of the CBS inhibitor aminooxyacetic acid (AOAA) in human colorectal cancer, in a translationally relevant animal model utilizing patient-derived xenografts (PDTX) in combination with current clinical anticancer therapeutic agents. Taken together, the current project will utilize human colonic cancer tissues, human colonic cancer cell lines and tumor-bearing mice subjected to heterotopic transplantation of human colon cancer lines or patient-derived xenografts. To address the role of CBS, a combination of genetic (CBS silencing) and pharmacological (CBS inhibitors) approaches will be used. Outcome variables will include parameters of bioenergetics (oxidative phosphorylation, mitochondrial electron transport, glycolysis, GAPDH activity), cell proliferation, cell growth (including activation of pr-inflammatory/pro-growth kinase pathways), tumor cell migration, tumor cell invasion, angiogenesis and metastasis in vitro and in vivo. The role of sulfhydration (a specific H2S-mediated posttranslational protein modification) will be also explored on relevant protein targets (GAPDH, PTEN, PI3K). The final, translational aim will contain studies of potential therapeutic relevance, by testing the anticancer effect of CBS inhibitors, in summary, the current project entails a comprehensive approach to test the importance of CBS/H2S a novel pathway in colorectal cancer, and incorporates early translational work to explore its potential utility as a target for anticancer therapy.